Biological Characteristics Of Human Umbilical Cord-derived Mesenchymal Stem Cells

BackgroundMesenchymal stem cells(MSCs)are a kind of pluripotent stem cells with self-renewal and multi-lineage differentiation potential.Depending on its powerful paracrine function,MSCs can not only promote the repair and regeneration of injured tissue,but also inhibit the injury of inflammatory reaction and maintain tissue homeostasis through their excellent immunoregulatory ability.In addition,MSCs can also promote the homing,proliferation and differentiation of hematopoietic stem cells.Besides,MSCs are widely available,they can be obtained from many tissues and easy to isolate and expand in vitro with few ethical or safety issues and low immunogenicity.A variety of unique advantages make them rapidly become a research hotspot.With the widespread development of related clinical trials,MSCs have shown great promise in regenerative medicine and the treatment of many diseases.Given the fact that human umbilical cord-derived mesenchymal stem cells(hUCMSCs)are easier to obtain and expand and have fewer ethical issues,many clinical trials are currently using hUC-MSCs alone or in combination with other drugs to treat severe degenerative and inflammatory diseases,including Crohn’s disease and graft-versus-host disease,and have successively concluded that hUC-MSCs are safe and effective.However,inconsistent results in existing studies still exist,making the clinical transformation of MSCs difficult.The heterogeneity of MSCs may be one of the reasons for this situation.It has been shown that MSCs from different donors,tissues,passages,clones,and even different cells of the same clone may have heterogeneity,while heterogeneous MSCs may exert different efficacy against the same disease.Therefore,identifying subpopulations of MSCs and analyzing the differences between different subpopulations are crucial to fully understand the function of MSCs and apply them in the clinical setting.In this paper,we investigated the heterogeneity of different passages of hUC-MSCs after in vitro amplification using high-throughput antibody microarray quantification,transcriptome sequencing(RNA-seq)and single-cell transcriptome sequencing(scRNAseq)technologies,in order to grasp the effects of in vitro culture on the biological characteristics of hUC-MSCs and promote the process of MSCs standardization.Methods1.The cultural supernatants of the 4th,6th,10th and 15th passages(P4,P6,P10 and P15)of hUC-MSCs were obtained,and the expression levels of 80 proteins were measured by high-throughput antibody microarray quantitative techniques,including human growth factor array(QAH-GF-1)and human inflammatory factor array(QAH-INF-3)kits.Concentrations of the proteins with the best confidence ratio of 50%-100%were set as reliable values for the quality control.Clustering heatmap was drawn for screening out the proteins that regularly increased or decreased with continuous passaging in vitro,and oneway ANOVA analysis was performed to select differentially expressed proteins with P<0.05 for further GO/KEGG enrichment analysis.2.hUC-MSCs of different passages were collected and analyzed by RNA-seq and 10×scRNA-seq.The differences of hUC-MSCs in different passages were compared to find differentially expressed genes.3.The differential expression of TNFRSF1 1B gene in different passages of hUC-MSCs was verified by quantitative real-time polymerase chain reaction(RT-qPCR)and ELISA assays.The differential expression of p53 gene in different passages was detected by RTqPCR and western blot(WB)assays.Results1.(1)The secretory proteins in the cultural supernatants of P4,P6,P10 and P15 hUCMSCs were quantified by high-throughput antibody microarray quantitative technology,and there were 39 proteins that were detected with satisfactory quality control results.The clustering analysis of the quality control-eligible proteins showed that with the increase of in vitro amplification,the expression levels of 16 proteins including IL-6,IL-11,IL-13,IL-17,M-CSF,GM-CSF,TNFR2,Eotaxin-1,MIP-1β,EGFR,SCFR,GDNF,1-309,GDF15,MCP-1 and IL-la exhibited a gradually down-regulated trend,while that of IL-7 was gradually up-regulated.Differences among the protein expression levels of IL-6,IL-11,IL-13,IL-17,M-CSF,GM-CSF,TNFR2,Eotaxin-1,MIP-1β,EGFR,SCFR,GDNF,1-309,GDF-15 and MCP-1 in the cultural supernatants of P4,P6,P10 and P15 hUC-MSCs were statistically significant(P<0.05),while there was no significant difference in the expressions of IL-1α and IL-7(P>0.05).The expression levels of IL-6,IL-11,IL-13,IL17,M-CSF,GM-CSF,TNFR2,Eotaxin-1,MIP-1β,EGFR,SCFR,GDNF,1-309,GDF-15 and MCP-1 in the supernatant of P4 hUC-MSCs were higher than those of P15 hUC-MSCs(P<0.05),and the expression levels of IL-6,IL-11,IL-13,IL-17,M-CSF,TNFR2,MIP1β,EGFR,GDNF,I-309,GDF-15 and MCP-1 in the supernatant of P10 hUC-MSCs were higher than those of P10 hUC-MSCs(P<0.05).The expression levels of IL-6,IL-11,IL13,M-CSF,TNFR2,MIP-1β,EGFR,GDNF and I-309 were higher than those of P6 hUCMSCs(P<0.05).The expression levels of IL-6,IL-11,IL-13,GM-CSF,TNFR2,Eotaxin1,EGFR,SCFR,GDNF and I-309 in the supernatant of P6 hUC-MSCs were higher than those of P15 hUC-MSCs(P<0.05),and the expression levels of TNFR2 and GDNF were higher than those of P10 hUC-MSCs(P<0.05).The expression levels of IL-6,IL-11,IL13,GM-CSF,GDNF and I-309 in the supernatant of P10 hUC-MSCs were higher than those of P15 hUC-MSCs(P<0.05).(2)GO enrichment analysis showed that the 15 differentially expressed proteins were mainly enriched in items such as signaling receptor activator activity,cytokine-mediated signaling pathway,positive regulation of cell migration and growth factor activity.KEGG pathway enrichment analysis showed that the 15 differentially expressed proteins were highly enriched in pathways such as cytokinecytokine receptor interaction,viral protein interaction with cytokine and cytokine receptor,rheumatoid arthritis and IL-17,TNF and JAK-STAT signaling pathway.2.(1)RNA-seq results showed that 1239 differentially expressed genes were screened in P6 and P15 hUC-MSCs.Compared with P15 hUC-MSCs,553 genes were down-regulated and 686 genes were up-regulated in P6 hUC-MSCs.TNFRSF11B,TXNRD1 and TUBA1B highly expressed in P15 hUC-MSCs,while LUM,VCAN and SERPINE2 lowly expressed.Then GO functional enrichment was performed and the molecular functions of differentially expressed genes were found to be enriched in the processes of receptor ligand activity,signaling receptor activator activity and ligand-gated ion channel activity.(2)The cells were divided into 10 clusters by dimensionality reduction analysis after scRNAseq,among which 4 clusters were mainly related to cell proliferation,DNA repair and stemness of MSCs.Three of the above 4 clusters were dominated by G2/M and S-phase cells.(3)The proportion of cells from different passages in each subset varied,and the above four functional subsets are mainly composed of P15 hUC-MSCs.Similar to RNAseq results,TXNRD1,TUBA1B,TNFRSF11B and IGFBP5 highly expressed in P15 hUCMSCs,while VCAN,SERPINE2,PDGFRA and LUM lowly expressed at the single-cell level.3.Compared with the lower passage,the expression of TNFRSF11B gene was significantly higher in P15 hUC-MSCs,while the expression of p53 was lower in P15 hUC-MSCs.Conclusions1.Continuous passaging of hUC-MSCs in vitro could result in phenotypic changes of the secretory proteins,as evidenced by diminished capacities of growth,differentiation,repair,immunoregulation and chemotaxis while increased pro-inflammatory and senescent phenotypes in higher passage of hUC-MSCs.2.The composition of different clusters of hUC-MSCs has obvious heterogeneity of passages after continuous amplification in vitro.A functional subgroup related to the proliferation,repair and stemness of hUC-MSCs was found.This functional subgroup is mainly composed of higher-passaged hUC-MSCs.Combined with the above multi-omics results,corresponding functional marker genes also transcripted in higher-passaged hUCMSCs,but the final function is still not as good as that in lower-passaged cells,indicating that the protein expression of these genes in higher-passaged hUC-MSCs has been affected.3.Referring to previous studies found that p53 gene plays an important role in regulation of osteogenesis and osteoblast differentiation,it is speculated that p53 may negatively regulate the expression of TNFRSF11B in hUC-MSCs.BackgroundAllogeneic hematopoietic stem cell transplantation(allo-HSCT)is an effective method to treat a variety of malignant hematological diseases,and it is also the only way to cure.However,graft-versus-host disease(GvHD)limits the wide application of HSCT.GvHD is a common complication and cause of death after HSCT,and it is also the major factor affecting the success of allo-HSCT.GvHD is a systemic disease caused by the recognition of host cell surface antigens by T lymphocytes in donor grafts,leading to multiorgan damage,which means that T lymphocytes not only play a key role in both immune reconstitution and anti-leukemia after hematopoietic stem cell transplantation,but can also cause GvHD.The current means to prevent and treat GvHD are mainly to down-regulate the effect of donor T lymphocytes,such as using glucocorticoids,immunosuppressants,and monoclonal antibodies,but at the same time,it can significantly reduce the immunity of host,resulting in an increased risk of lethal infection and tumor recurrence.Therefore;,how to effectively combat GvHD while preserving the anti-infective and anti-tumor effects is an urgent issue when performing allo-HSCT.Mesenchymal stem cells(MSCs),as "living" cell-based drugs in the field of regenerative medicine,have powerful regulatory effects on immune and inflammatory responses and have promising applications in some autoimmune and inflammatory diseases such as GvHD,rheumatoid arthritis,systemic lupus erythematosus and diabetes.In view of the possibility of MSCs preventing GvHD after allo-HSCT,this study aims to establish a stable and typical animal model of acute graft-versus-host disease(aGvHD)and give hMSCs injection to observe the occurrence of aGvHD,detect the changes of blood cells after transplantation,and use single-cell transcriptome sequencing(scRNA-seq)technique to analyze the transcriptome characteristics of peripheral blood and bone marrow mononuclear cells of mice after hMSCs treatment,so as to understand the role of hMSCs in preventing aGvHD and provide experimental basis for clinical application of hMSCs.Methods1.Bone marrow and spleen cells from donor mice were collected,mixed in a certain ration and injected into recipient mice irradiated with lethal dose to construct aGvHD mouse model.Different passages and different doses of hMSCs were infused 24 hours after transplantation.2.The clinical manifestations,clinical scores,survival time,body weight and hematological changes of mice were monitored to and the passage and dose of hMSCs with better curative effect were compared.3.Bone marrow and peripheral blood of mice in myeloablative group,transplantation group and multiple hMSCs treatment groups were collected,mononuclear cells were isolated and scRNA-seq was performed on 10× Genomics platform.Results1.The aGvHD mouse model was successfully constructed.2.(1)Clinical manifestations:The condition of mice in the transplantation group continued to deteriorate and showed weight loss,piloerection,arching of the back,perianal redness and swelling.loose stools,hair removal and reduced mobility with the extension of transplantation time.Compared with the transplantation group,the condition of mice in hMSCs A high-dose group and hMSCs B low-dose group was significantly better.(2)Clinical scores:Compared with the myeloablative group,the clinical score of mice in the transplantation group decreased significantly(P<0.01).Compared with the transplantation group.the clinical scores of mice in hMSCs A high-dose group increased signiflcantly on the 4th day(P<0.05)and decreased signiflcantly from the 14th day to the 23rd day(P<0.01)after administration,while that in low-dose groups of hMSCs A and hRSCs B increased significantly from the 7th day to the 10th day(P<0.05)and decreased significantly on the 14th day(P<0.01),and that in the high-dose groups of hMSCs B and hMSCs C increased significantly on the 7th day(P<0.05)and decreased significantly on the 14th day(P<0.01)after administration.The clinical scores of mice in hMSCs C lowdose group decreased significantly on the 14th day after administration(P<0.01).(3)Survival:Compared with the myeloablative group,the survival of mice in the transplantation group was significantly prolonged(P<0.01),and the life extension rate was 26.4%.Compared with the transplantation group,there was no significant difference in survival among the other groups.(4)Body weight:Compared with the myeloablative group,the body weight of mice in the transplantation group decreased significantly on the 7th(P<0.01)day and increased significantly on the 10th day(P<0.01)after administration.Compared with the transplantation group.the body weight of mice in hMSCs A high-dose group and hMSCs B low-dose group tended to increase from the 21st to the 23rd day after administration,but there was no significant difference statistically(P>0.05).(5)Hematology:After 7 days of administration,the number of leukocytes,monocytes and neutrophils in the transplantation group was significantly higher than that in the myeloablative group(P<0.01,P<0.05,P<0.01).Compared with the transplantation group,the number of leukocytes,lymphocytes,monocytes and neutrophils in hMSCs A low-dose group increased significantly(P<0.05),and that in two groups of hMSCs B and hMSCs C high-dose group increased significantly(P<0.01),The number of neutrophils in hMSCs C low-dose group increased significantly(P<0.01).After 14 days of administration,compared with the transplantation group,the number of lymphocytes and monocytes in hMSCs A high-dose group increased significantly(P<0.05).The number of lymphocytes in two groups of hMSCs B both increased significantly(P<0.05).After 23 days of administration,there was no significant difference in the number of blood cells in each administration group compared with the transplantation group(P>0.05).Finally,the serum levels of IL-6 in mice of hMSCs A high-dose and hMSCs B low-dose groups were significantly decreased compared with the transplantation group(P<0.05,P<0.01).3.Using 10× Genomics platform,scRNA-seq was performed on mononuclear cells of bone marrow and peripheral blood from myeloablative,transplantation group,hMSCs A and hMSCs B treatment groups.The gene expression profiles of different groups at singlecell level were described,and there were differences in gene expression among groups.Through enrichment analysis,it was found that differentially expressed genes were mainly enriched in the process of mitosis in bone marrow,while in peripheral blood they were mainly enriched in the processes of mononuclear cells,lymphocytes and T cells differentiation,coagulation,homeostasis and wound healing,as well as pathways such as T cell receptor signaling pathway,antigen processing and presentation and hematopoietic cell lineage.Conclusions1.hMSCs A high-dose group and hMSCs B low-dose group can significantly improve the survival quality of aGvHD mice,reduce the serum levels of inflammatory factors such as IL-6,inhibit the rejection caused by bone marrow transplantation and prolong the survival time.2.hMSCs may be able to assist the graft to reconstitute immunity in the early stage after bone marrow transplantation.3.hMSCs treatment can affect the proliferation of mononuclear cells in bone marrow and peripheral blood after transplantation.We believe that the proliferative cells in bone marrow are mainly proerythrocytes,hematopoietic precursor cells and granulocytopoietic cells,and that in peripheral blood are neutrophils,NK cells and B cells.